Nucleic acids are biological molecules essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from nucleotides. Nucleic acids function in encoding, transmitting and expressing genetic information. Studies of nucleic acids are a critical part of modern biological and medical research, which form a foundation for genomics as well as the biotechnology and pharmaceutical industries.
Feeling the constraints set by the very limited number of naturally occurring nucleotides in standard nucleic acids, scientists have for years attempted to introduce unnatural base pair systems in an effort to expand the genetic code. One such unnatural base pair is the so-called “Z-P” system developed by Steven Benner and collaborators in which the synthetic nucleotide Z can pair with the synthetic nucleotide P via 3 hydrogen bonds. (Kim, et al. 2014 J. Org. Chem. 79 (7) 3194-3199; Yang, et al. 2013 Anal. Chem. 85 (9) 4705-4712, Kim, et al. 2012 J. Org. Chem. 77 (7) 3664-3669; Yang, et al. 2011 J. Am. Chem. Soc. 133 (38) 15105-15112; Chen, et al. 2011 Nucleic Acids Research 39(9) 3949-3961; Yang, et al. 2010 Angewandte Chemie, International Edition 49(1) 177-180, S177/1-S177/16; Yang, et al. 2007 Nucleic Acids Research 35(13) 4238-4249; Yang, et al. 2006 Nucleic Acids Research 34(21) 6095-6101; Hutter, et al. 2003 J. Org. Chem. 68(25) 9839-9842; WO 2009/154733 A2; U.S. Pat. No. 8,389,703 B1; U.S. Pat. No. 8,586,303 B1.)
Among the important requirements for a proposed unnatural base pair to serve as components of the expanded genetic code, it must allow for easy, clean incorporation of the unnatural nucleotides into DNA and RNA molecules by conventional chemical DNA and RNA synthesis. Oligonucleotide synthesis of desired length and sequence with defined chemistry is a fundamental criterion in both laboratory research and applications in molecular biology and medicine.
During oligonucleotide synthesis, in order to prevent undesired side reactions, select functional groups present in nucleosides have to be rendered unreactive or “protected” by attaching protecting groups. The protecting groups are removed to yield the desired oligonucleotides at the completion of a particular synthetic step or after the assembly of an oligonucleotide.
One of the few reported protecting groups used for the Z nucleotide base when making synthetic DNA (or RNA) incorporating Z nucleotide is a para-nitro phenylethyl (NPE) protecting group (U.S. Pat. No. 8,389,703 B2.) However, experiments to make RNA containing Z nucleotides protected with NPE have shown that this protecting group is far from ideal. In fact, significant damage to oligonucleotide product occurs during deprotection along with undesired modification of the Z nucleotide itself. As a result, currently there is no viable protection strategy for efficient synthesis of oligonucleotides incorporating Z nucleotide, which severely restricts the utility of the Z-P unnatural base pair.
Thus, there is an urgent need to develop novel protecting groups and methodologies to enable easy chemical incorporation of unnatural nucleotides, in particular Z nucleotides, into synthesized long oligonucleotides.